The present invention relates to a recombinant lysophosphatidic acid phosphatase (LPA phosphatase). More particularly, the present invention relates to a DNA encoding an LPA phosphatase, a protein encoded by the DNA, an expression vector carrying the DNA, a transformant harboring the expression vector, a process for preparing the protein, an antibody against the protein, an antisense DNA or RNA complementary to the DNA, an oligonucleotide probe or primer capable of specifically hybridizing to the DNA, a method for determination of LPA using a recombinant LPA phosphatase, a determination reagent and a kit for diagnosis.
Lysophosphatidic acid (LPA) is a naturally-occurring phospholipid having the simplest chemical structure, and it has been known to exhibit a growth factor-like activity against the fibroblasts [Moolenaar, W. H. et al., (1992) Rev. Physiol. Biochem. Pharmacol. 119, 47-65; Moolenaar, W. H., (1995) J. Biol. Chem. 270, 12949-12952; Moolenaar, W. H. et al., (1997) Curr. Opinion Cell Biol. 9, 168-173]. The LPA is rapidly produced and released as a product of the coagulation process of blood from the activated platelets. Therefore, it is suggested that the LPA has a kind of a role in healing of a wound and regeneration. Also, there is clarified that the LPA induces rapid retraction of the axon and transient formation of spheres of cell bodies in neurocytes. These biological activities are thought to be caused by LPA receptor conjugated with G protein [Moolenaar, W. H. et al., (1997) Curr. Opinion Cell Biol. 9, 168-173], and a cDNA encoding two deduced LPA receptors has been isolated [Hecht, J. H. et al. (1996) J. Cell Biol. 135, 1071-1083; Guo, Z. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14367-14372].
In addition, the LPA possesses various biological activities such as promotion of cancer cell invasion, cell adhesion, suppression of apoptosis, and chemotaxis. Moreover, there has been reported that its amount present in sera is high in ovarian cancer and other gynecologic malignancies [Xu, Y. et al. (1998) JAMA 280, 719-723], and it is expected to also serve as a marker for early detection of cancers.
Regarding the production of the LPA, there has been known that the LPA is synthesized by generation from monoacylglycerol by monoacylglycerol kinase, generation from phosphatidic acid by phospholipase A1 (PLA1) or phospholipase A2 (PLA2), and the like. However, since the LPAs produced in response to various stimuli take place at a slightly delayed stage as compared to the generation of phosphatidic acid (PA) [Gait, F. et al. (1997) FEBS Lett. 410, 54-58], it is thought that those ascribed to the latter pathway are dominant.
However, the LPA in the biosynthesis system is immediately converted to phosphatidic acid, so that it is thought that most of the cases where the LPA acts as a bioactive substance is not made from the synthesis system but as a product of a phospholipid degradation system. Regarding the hydrolytic pathways of the LPA, there are possibly three pathways, namely pathways with LPA phospholipase A, LPA phosphatase and LPA acyltransferase [Gait, F. et al., (1997) FEBS Lett. 410, 54-58; Eberhardt, C. et al., (1997) J. Biol. Chem. 272, 20299-20305]. Since the LPA is a bioactive lipid, it is thought that the exclusion of the LPA by the above enzyme plays an extremely important role in termination of the signal.
Regarding LPA phospholipase A, one purified from rat brain has been known, which is a membrane bound enzyme of a size of 80 kDa, and hydrolyzes LPA but does not hydrolyze other lysophospholipids [Thompson, F. J. and Clark M. A., (1994) Biochem. J. 300, 457-461].
Regarding the LPA phosphatase, there has been reported the presence of ecto-(lyso)phosphatidic acid phosphatase that also hydrolyzes PA in PAM212 mouse keratinocytes [Xie, M. and Low, M. G., (1994) Archives Biochemistry Biophysics 312, 254-259]. Until recently, a membrane bound PA phosphatase which is relatively PA-specific but also exhibits a weak activity for LPA has been purified from porcine thymus [Kanoh, H. et al., (1992) J. Biol. Chem. 267, 25309-25314; Kai, M. et al., (1996) J. Biol. Chem. 271, 18931-18938]. In addition, a membrane bound PA phosphatase, which also hydrolyzes LPA, ceramide 1-phosphate and sphingosine 1-phosphate, is purified from rat liver [Waggoner, D. W. et al., (1995) J. Biol. Chem. 270, 19422-19429; Waggoner, D. W. et al., (1996) J. Biol. Chem. 271, 16506-16509]. However, there has not yet been known LPA-specific phosphatase at present.
Further, regarding the method for determination of LPA, there has been known a method comprising extracting lipid components from a sample, separating LPA from other lipid components by thin layer chromatography, and thereafter determining the resulting LPA by gas chromatography as a methyl ester of a fatty acid via transmethylation reaction [Xu, Y. et al. (1998) JAMA280, 719-723]. However, this method requires complicated procedures, so that it has a defect that a length of time period is required for assay of a large number of samples.
The present invention has been accomplished in view of the above prior art, and an object of the present invention is to provide a recombinant LPA phosphatase capable of specifically hydrolyzing LPA, which is useful for elucidation of the metabolic pathway for LPA, and also for diagnosis and treatment for various diseases with which LPA is associated; a method capable of simply and inexpensively determining LPA associated with various diseases; a determination reagent suitable for the method; a kit suitable for diagnosis of a disease with which LPA is associated, and the like.
The present inventors have confirmed the presence of the LPA phosphatase activity in bovine brain, and purified the LPA phosphatase using bovine brain as a raw material, and further proceeded with the study. As a result, they have succeeded in cloning human LPA phosphatase gene, and the present invention has been perfected thereby.
The gist of the present invention relates to:
[1] a DNA encoding a lysophosphatidic acid phosphatase, wherein the DNA is selected from the group consisting of:
(a) a DNA encoding a peptide comprising the amino acid sequence of SEQ ID NO: 1;
(b) a DNA encoding a peptide comprising an amino acid sequence resulting from deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1;
(c) a DNA comprising the nucleotide sequence of SEQ ID NO: 2;
(d) a DNA comprising a nucleotide sequence resulting from deletion, substitution, insertion or addition of one or more bases in the nucleotide sequence of SEQ ID NO: 2;
(e) a DNA capable of hybridizing to the DNA of any one of the above (a) to (d), under stringent conditions;
[2] a protein encoded by the DNA of item [1] above;
[3] an expression vector carrying the DNA of item [1] above;
[4] a transformant harboring the expression vector of item [3] above;
[5] a method for producing a recombinant lysophosphatidic acid phosphatase, comprising the step of culturing the transformant of item [4] above under conditions capable of expressing a protein from the expression vector of item [3] above;
[6] an antibody or a fragment thereof, capable of specifically binding to the protein of item [2] above;
[7] an antisense DNA or antisense RNA comprising 8 bases or more, having a sequence complementary to the DNA of item [1] above;
[8] a probe or primer, capable of specifically hybridizing to the DNA of item [1] above;
[9] a method for determination of LPA, comprising the step of mixing the protein of item [2] above with a sample to be tested;
[10] the method for determination of LPA according to item [9] above, wherein the presence or absence of a product resulting from hydrolysation of lysophosphatidic acid by the protein of item [2] above is used as an index of the presence or absence of lysophosphatidic acid;
[11] the method for determination of LPA according to item [10] above, wherein phosphoric acid or monoacyl glycerol is determined as the hydrolysate of lysophosphatidic acid;
[12] a determination reagent for LPA, comprising the protein of item [2] above;
[13] the determination reagent for LPA according to item [12], further comprising a reagent for determining phosphoric acid or monoacyl glycerol; and
[14] a kit for diagnosing a disease in which LPA is involved, comprising the determination reagent for LPA of item [12] or [13] above.